Ewing's sarcoma is an aggressive pediatric cancer of the bone and soft tissue. Patients that present with metastasis and patients who relapse have especially poor outcomes from this disease. Ewing's sarcoma is driven by a chromosomal translocation that produces the EWS/Fli1 fusion protein. We have identified the developmental protein, Eya3, as a downstream target of EWS/Fli1. Eya3 is important for DNA repair and is also found as part of a bipartite transcription factor complex with the Six family of homeobox proteins. The Eya/Six complex is important for development, but when inapropriately expresed after development is complete, promotes many oncogenic properties in other cancers, including proliferation, survival, and metastasis. However, the role of this complex in pediatric tumors, specificaly Ewing's sarcoma, has never before been examined. Similar to what is observed in other human tumors, inhibition of Eya3 in Ewing's sarcoma cells has a modest effect on proliferation and survival. However, we have discovered a novel role for Eya3 in Ewing's sarcoma, where Eya3 knockdown significantly increases Ewing's sarcoma cell chemosensitivity. This may be the result of the recently described ability of Eya3 to facilitate efficient DNA repair Additionally, since tumor-initiating cells (TIC) are implicated in treatment-resistant (chemoresistant) cancers, and since the binding partner of Eya3, Six1, increases TIC populations in breast cancer, we further examined whether Eya3 may modulate a TIC population in Ewing's sarcoma. Indeed, Eya3 knockdown decreases the tumor-initiating cell population. This data suggests that Eya3 may play an important role in mediating Ewing's sarcoma phenotypes associated with recurrent disease through multiple, and possibly inter- related, mechanisms. Studies outlined in this proposal aim to better understand the role of Eya3 in phenotypes associated with Ewing's sarcoma relapse, in an attempt to identify novel new drug targets that when inhibited, may reduce the poor clinical outcomes associated with Ewing's sarcoma. Studies performed will allow us to determine the mechanism of EWS/Fli1 regulation of Eya3 and then go on to further evaluate the role of Eya3 in modulating tumor-initiating cell populations and in chemoresistance. Eya3 has a unique tyrosine phophatase domain, as well as a transcriptional activation domain required to drive transcription along with its oncogenic partner, Six1. Work within this proposal will allow us to determine which activities of Eya3 are important for its roles in Ewing's sarcoma, so that we can better guide our targeting of Eya3 with small molecule inhibitors.